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Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.


physiology, Synaptic Vesicles, Synaptic Transmission, Recombinant Fusion Proteins, Rats, Mutagenesis, Molecular Sequence Data, genetics, Luminescent Proteins, Indicators and Reagents, Hydrogen-Ion Concentration, Humans, cytology, Hippocampus, HeLa Cells, Green Fluorescent Proteins, Fluorescent Dyes, Exocytosis, Cytoplasmic Granules, Cells, Cultured, Animals

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      In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors. Here we describe sensors for secretion and neurotransmission that fulfil these criteria. We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.

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